Two Mutant Forms of Human Insulin. Structural Consequences of the Substitution of Invariant B24- or B25-Phenylalanine by Leucine

Abstract

In 1979 the first abnormal human insulin was discovered. With the minute samples from the patient a Phe .fwdarw. Leu replacement could be established in either position of B24 or B25. For the unequivocal localization of the substitution both the Leu analogs had to be prepared by semisynthesis. While another laboratory did this with the sequence of porcine insulin, here true analogs of human insulin are studied. The structural consequences of the substitutions are investigated. Human insulin obtained by total synthesis served as a reference. Its CD [circular dichroism] spectral properties are documented. According to the substantial deviations of the CD spectrum of [LeuB24]insulin, the introduction of the new side-chain forces conformational changes to occur not only in its immediate sourrounding but also in the peptide chain. The failure to give the typical CD spectral response to variations of protein and Zn concentration indicates that the ability to form quaternary structure is impaired. Though dimerization was confirmed by gel chromatography to be largely reduced, interactions normally responsible for the increase in tyrosine-CD with association are weakened. [LeuB25]insulin does exhibit all CD spectral effects characteristic of the native hormone, though quantitatively somewhat reduced. The CD spectroscopic results are in full agreement with the computergraphic analysis of the sterical consequences of the substitutions. For B24-leucine an acceptable packing without movements of the mainchain and/or B15-leucine and without affecting dimerization is impossible; B25-leucine can be accommodated without causing bad contacts either in the monomer or in the dimer. The structural results do not explain why [LeuB25]insulin should have a lower biological activity than the B24 analog, 2.1 .+-. 0.3% vs. 20.9 .+-. 2.8%, in the fat cell test. They suggest an important but not critical stereospecific role for the B25-phenylalanine in binding.